The use of inhibitors of the sodium-hydrogen exchanger for preparing a medicament for preventing age-related organ dysfunction and age-related disorders, and for prolonging life.
The invention describes the use of inhibitors of the cellular sodium-hydrogen exchanger in human and veterinary medicine for preventing age-related functional disturbances and dysfunctional changes of body organs and for preventing age-related disorders, and for prolonging life while preserving an improved quality of life.
Within the last years, inhibitors of the sodium-hydrogen exchanger (NHE) have been characterized in numerous preclinical studies as substances which, in cases of hypoperfusion of the heart, are suitable in a superior manner for protecting the heart tissue at risk from the acute onset of the ischemia event from destruction. The protection of heart tissue by NHE inhibitors embraces all types of damage caused by the hypoperfusion, from cardiac arrhythmia to hypercontraction of the heart muscle and temporary loss of function to necrosis of heart tissue and associated permanent damage.
The mechanism of action of the NHE inhibitors, which is important in acute ischemic events, consists in that they reduce the increased influx of sodium ions which takes place in acute hypoperfused tissue by activation of NHE owing to intracellular acidification. This delays the situation of a sodium overload of the tissue. Since in heart tissue sodium and calcium ion transport are coupled with each other, this prevents the life-threatening calcium overload of the heart cells.
In the same manner as at the heart, most of the patents cited here also describe protection of the central nervous system under the influence of NHE inhibitors, and such active compounds protect the CNS similarly to the heart against acute ischemic states. These states are caused by an acute hypoperfusion and thus by insufficient supply of nutrients, oxygen, minerals, etc. Such ischemic damage of the CNS is particularly pronounced in the case of central infarcts, such as stroke.
Consequently, in accordance with expectations, in the case of normal healthy perfusion it was of course not possible to observe any protective effects of NHE inhibitors against these acute events, since there was no acute onset of ischemic tissue damage of the heart or the CNS.
Thus, it was surprising that NHE inhibitors, in addition to the protective effects against acute ischemic events and the subsequent likewise acutely stressing reperfusion events, also have direct therapeutically utilizable effects against disorders and disturbances of the entire organism of mammals which are connected with the manifestations of the chronic aging process and which are independent of acute states of hypoperfusion and which occur under normal non-ischemic conditions. These pathological age-related manifestations such as diseases, infirmity and death, which occur during the long period of aging and which are now accessible to treatment with NHE inhibitors are disorders and disturbances which are caused mainly by age-related changes of vital organs and their function and which become more and more important in the aging organism. NHE inhibitors such as, for example, cariporide act on such age-related disorders and disturbances of organs and their functions by a cascade of primary and secondary mechanisms whose mechanism has not yet been elucidated completely. It was therefore not possible to expect or predict a life-prolonging and antiaging effect for the NHE inhibitors, not to mention that such pronounced and strong effects have, according to our knowledge, not yet been shown in a comparable manner for any of the classes of active compounds known to date. It is true that recently a life-prolonging effect of ACE inhibitors, in particular under ramipril treatment, has been reported (Linz W, Jessen T, Becker RHA, Schxc3x6lkens BA, Wiemer G. Long-term ACE inhibition doubles lifespan of hypertensive rats. Circulation. 1997; 96: 3164-3172). However, this life-prolonging effect only relates to hypertensive rats and organisms which, owing to their high blood pressure, have a reduced life expectancy compared with normotensive rats and organisms, respectively. Thus, the life-prolonging effect of ACE inhibitors relates only to animals suffering from high blood pressure and can, in the best case, only achieve the lifespan of a normotensive organism. Since at least a considerable part of the activity of the ACE inhibitors is caused by their hypotensive effect on individuals suffering from high blood pressure, the lack of a life-prolonging effect of ACE inhibitors on normotensive individuals came as no surprise. Likewise, ACE inhibitors did not show to a comparable extent an inhibiting action on age-related organ damage or the onset of cancer induced by the aging process.
Disorders associated with an age-related functional disturbance or with age-related manifestations of wear and tear of organs are, for example, the insufficient responsiveness and reactivity of the blood vessels to contraction and relaxation reactions. This age-related decrease of vessel reactivity on constrictory and relaxing stimuli, which are an essential process of the cardiovascular system and thus of life and health, can be eliminated or reduced significantly by NHE inhibitors. An important function and a measure for the maintenance of vessel reactivity is the blockage or delay of age-related progressive endothelial dysfunction which can be eliminated by NHE inhibitors in a highly significant manner.
An example of another measurement which characterizes the aging process is the reduction of the contractility of the heart and the decreased adaptation of the heart to a required pumping performance of the heart. This reduced ability of the heart to perform, as a consequence of the aging process, is in most cases associated with heart dysfunction caused, inter alia, by incorporation of connective tissue into heart tissue. This incorporation of connective tissue is characterized by an increase in the weight of the heart, by an increased heart size and by impaired heart function. It was surprising that such an aging of the organ""s heart could be inhibited almost completely.
Whereas the earlier patents and patent applications claimed the treatment of various forms of cancerous diseases which have already become manifest, it was now extremely surprising that it is not only possible to treat a manifest cancerous disorder by inhibition of proliferation, but that it was also possible to prevent and delay in a highly significant manner the age-related frequency of the occurrence of cancer by using NHE inhibitors. Particularly noteworthy is the finding that age-related disorders of all organs and not only of certain forms of cancer can be prevented, or delayed in a highly significant manner.
What is found now is not only a delay of the onset of age-related disorders of all organs examined, including heart, vessels, liver, etc., delayed in a highly significant manner for more than the statistical norm, and a highly significant delay of age-related cancer. Rather, life is surprisingly prolonged to an extent which hitherto has not been achieved by any other group of medicaments or any natural substances.
This unique effect of the NHE inhibitors makes it also possible, in addition to using the active compound on its own in humans and animals, to combine these NHE inhibitors with other gerontologically applied principles of action, measures, substances and natural substances which are based on a different mechanism of action. Such classes of active compounds used in gerontological therapy are: in particular vitamins and antioxidants. Since there is a correlation between caloric stress or food intake and the aging process, a combination with dietary measures, for example with appetite suppressants, is possible. Also feasible is a combination with hypotensive medicaments, such as ACE inhibitors, angiotensin receptor antagonists, diuretics, Ca2+ antagonists, etc., or with medicaments which have a normalizing action on the metabolism, such as cholesterol-lowering substances.
Studies
The effect of a life-long treatment of young normotensive Wistar-Kyoto rats (WKY rats) with the sodium-hydrogen exchanger (xe2x80x9cNHExe2x80x9d) inhibitor cariporide was investigated. In addition to the primary end point, the lifespan, surrogate parameters were studied and measured, for example the hypertrophy of the left ventricle of the heart, heart function, metabolism and endothelial function. In addition, organs, such as heart, kidneys, skeletal muscles and eyes, were examined histomorphologically.
Methods and Results
Ninety one-month-old WKY rats were randomized into two groups of placebo-treated (n=45) and cariporide-treated (n=45) rats. Cariporide was administered via the feed (active compound content 0.3%).
Cariporide is the compound 
(see U.S. Pat. No. 5,292,755)
The surrogate parameters were determined after 30 months, at which time about 80% of the placebo group had already died. The life-long treatment with cariporide prolonged the life of the animals from 30 to 39 months. This prolongation of life correlated with a delayed onset of age-related cancer. Age-related hypertrophy of the heart, which occurred in placebo-treated animals, was prevented by cariporide. A significantly improved heart function in comparison with the placebo hearts was achieved by cariporide. The NHE inhibitor also improved age-related endothelial dysfunction significantly. Age-related morphological organ changes, such as the incorporation of connective tissue into the aging heart muscle, age-related tubulo-interstitial lesions of the kidney, muscular dystrophy and retina atrophy were reduced drastically or prevented completely by cariporide.
Result
The life-long treatment of normotensive WKY rats with the NHE inhibitor cariporide prolonged the lifespan of the animals from 30 to 39 months. This extension of the lifespan correlated with a prevention of age-related hypertrophy of the left ventricle, of cardiac and vascular dysfunction and of age-related changes of vital organs. In addition, the onset of all forms of age-related cancer was delayed significantly.
Methods
Animals
Male WKY rats were purchased from Mxc3x8llegrad Denmark. The animals (3 per cage) were kept under standardized conditions with respect to temperature, atmospheric humidity and light. The rats had free access to a standard diet (Altromin(copyright) maintenance diet 1320, sodium content 0.2%) and received drinking water ad libitum. All experiments were carried out in accordance with the German Law on the Protection of Animals.
Study Design
Ninety one-month-old animals were randomized into two groups of in each case 45 animals. After randomization, placebo and cariporide were administered to the rats via the drinking water and the feed, respectively, a proportion of 0.3% of the feed ensuring complete blockage of the NHE for 24 hours.
At intervals of 3 months, the body weight was determined and the blood pressure was measured using the tail plethysmography method. Events of death were registered after they had occurred.
Interim Analysis
The interim analysis was carried out after 80% of the placebo-treated animals had died; this was the case after 30 months. 10 animals of each group were selected at random and anesthetized (hexobarbital, 80 mgxc2x7kgxe2x88x921, i.p.). Blood samples were then taken, and the thoracic aortae and the hearts were prepared and biochemically and/or functionally examined (Linz et al., 1997).
Examination of the Function of the Isolated Heart
In accordance with the method described by Langendorff, the hearts were perfused with an oxygen-saturated (95% O2, xe2x88x925% CO2), non-circulating Krebs-Henseleit solution of the following composition (mmol/l): NaCl, 118; KCl, 4.7; CaCl2, 2.5; MgSO4, 1.6; NaHCO3, 24.9; KH2PO4, 1.2; glucose, 5.5; Na-pyruvate, 2.0, using a perfusion pressure of 60 mm Hg. The pressure in the left ventricle and the pulse frequency of the heart were measured using a balloon catheter. The coronary blood flow was determined using an electromagnetic flow gage. After a washing phase of 5 minutes, 1 ml of the coronary effluent was taken off for measuring the lactate concentrations and the lactate dehydrogenase and creatine kinase activities. The weights of the entire heart and the weights of the left and the right ventricle were then determined. The left ventricle was fixed in 10% strength neutral buffered formalin solution and embedded in paraffin for light-microscopic examination.
Vessel Function on Isolated Rings of the Thoracic Aorta
The aorta was introduced into Tyrode""s solution and freed from the surrounding fatty tissue. Segments of 2 mm were then sliced into rings, and these rings were introduced in a temperature-controlled (37xc2x0 C.) organ bath (10 ml) with Tyrode""s solution (composition in mol/l: NaCl 136.9; NaHCO3 11.9; KCl 2.7; CaCl2 0.5; MgCl2 2.0; NaHPO4 0.4; glucose 5.5; pH=7.4), and 95% O2/5% CO2 was bubbled through the solution. Each ring was mounted vertically between two fine steel clamps. The upper clamp was connected to a force sensor. The force signal was recorded using a computer-assisted biosignal analyzer. The aorta rings were suspended using a passive tension of 4.9 mN. After an adaptation period of one hour, the rings were contracted using potassium chloride (20 mmol/l). After a plateau had been reached, increasing concentrations of acetylcholine of 10xe2x88x928, 10xe2x88x927, 10xe2x88x926 and 10xe2x88x925 mol/l were added to the bath, triggering an endothelium dependent relaxation.
Statistical Analysis
All data are stated as meanxc2x1standard deviations. The survival data were analyzed in accordance with Kaplan-Meier, followed by a Mantel-Cox log-rank test. Furthermore, variance analysis (ANOVA) followed by a Student-Newman-Keuls test were employed. Null hypotheses were rejected at p less than 0.05.
Results
The body weight increased from 72xc2x13 g (1 month old) to 517xc2x119 g (30 months old) in the placebo-treated animals and to 519xc2x120 g in the cariporide-treated animals. In the placebo-treated WKY rats, the systolic blood pressure of 119xc2x14 mm Hg (mean of all animals at the age of 1 month) remained almost unchanged up to the age of 30 months, 120xc2x13 mm Hg. NHE inhibition with cariporide did not result in any significant change of the blood pressure in WKY rats (121xc2x12 mm Hg).
Cumulative Survival Rate
Age-related, the placebo-treated WKY rats began to die after about 19 months, and they had all died after the experiment had lasted 30 months. In contrast, the maximum lifespan of the animals was extended by cariporide treatment to 39 months, corresponding to a life extension of 30% (Table 1).
Interim Analysis of the Study after 30 Months
Due to the cariporide treatment, the weight of the left and right ventricle of the heart were significantly reduced (Table 2).
At 332xc2x16 mg/100 mg of body weight, the weights of the kidneys of the two groups did not show any significant difference between the groups, whereas the weights of the spleens of the animals which had been treated with cariporide were significantly below those of the placebo-treated group of WKY rats (303xc2x112 versus 255xc2x113 mg/100 g of BW; p less than 0.05).
Isolated Heart Preparations
Hearts of cariporide-treated WKY rats showed a significant increase in the pressure of the left ventricle and the contractile force (dP/dtmax) of the left ventricle, compared with hearts of placebo-treated WKY rats. Here, the pulse frequency of the hearts was unchanged, whereas the coronary blood flow under cariporide was significantly higher than that in placebo-treated rats (Table 3).
In the hearts of normotensive WKY rats, the long-term treatment with cariporide resulted in a reduction of the activities of cytosolic enzymes (creatine kinase, lactate dehydrogenase) and in a reduced release of lactate into the coronary effluvium. This surprising finding demonstrates the considerably worse metabolic situation of the aged rats without cariporide (Table 3).
Isolated Thoracic Aorta
The endothelium-dependent relaxation of aorta strips precontracted with potassium chloride was examined. Here, aorta strips of the 30-month-old placebo-treated WKY rats showed the known deterioration of the endothelium-dependent relaxation which is reduced in an age-related manner.
In the 30-month-old WKY rats which had been treated with cariporide, relaxation was significantly better (Table 4).
Histomorphological Results
Cariporide drastically reduced typically age-related histomorphological organ changes, such as the incorporation of connective tissue into the heart muscle, muscular dystrophy, tubulointerstitial damage of the kidney and retina atrophy. In addition, the livers of these rats were unchanged, compared with control animals.